JP4216076B2 - Drugs for the prevention and treatment of sexually transmitted diseases-I - Google Patents

Abstract

Use of a polylysine, polyamidoamine or polypropylenimine dendrimer having naphthyl disulphonate terminal groups as a topically applied agent in prophylaxis or treatment of sexually transmitted diseases.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to the prevention and treatment of sexually transmitted diseases, and in particular to the use of dendrimers having naphthyl disulfonate end groups as agents applied topically to the prevention and therapeutic treatment of these diseases. .
Background art

  The global incidence, morbidity, and mass death of sexually transmitted diseases (STD) caused by HIV, HSV, and other viral and microbial pathogens are estimated to be several hundred million worldwide. One method of controlling the transmission of STD is the use of topically applied female / male controlling vaginal or rectal fungicides that deactivate the associated pathogens. Consequently, the development of new, safe, topical bactericides for intravaginal or rectal use for the prevention and treatment of STD is an important target for new drug development.

International patent applications PCT / AU95 / 00350 (WO95 / 34595) and PCT / AU99 / 00763 (WO00 / 15240), the contents of which form part of the present invention, are a new class of multivalent drugs-dendrimers, polyanionic Alternatively, highly branched polymers having a constant envelope of polycationic surface groups are disclosed. It is known to exhibit a range of antiviral and antimicrobial activity with minimal toxicity. Unlike the small molecular structure of most antiviral agents, these dendrimers are one multivalent highly branched macromolecular compound that is contiguous to create a three-dimensional highly ordered polymer compound. Formed by iterative reaction sequences starting from the first core molecule with successive layers or steps of “generation” added. Dendrimers are characterized by the following characteristics:
i. An initiator core that has one or more reactive sites and is dot-like or significantly sized to affect the final topology of the dendrimer,
ii. A layer of branched repeating units attached to the initiator core,
iii. A functional end group (a moiety containing an anion or cation) optionally attached to the surface of the dendrimer via a linking group.

  These polymeric compounds are synthesized from monomeric building blocks having a large number of branch or tree-like structures, and the outer surface of the molecule has a large number of functional groups that are guided to recognition by biological receptors.

  The preparation of dendrimers is well known, for example US Pat. Nos. 4,289,872 and 4,410,688 (dendrimers based on layers of lysine units) and also US Pat. Nos. 4,507,460, 4,558,120, 4,568,737 and 4,587,329. (Dendrimers based on other units including polyamidoamines or PAMAM dendrimers).

式中Ｒは式ＩＶの基を表わす。

Summary of the Invention The present invention provides a compound of formula I, II, or III or a pharmaceutically acceptable salt thereof.

In which R represents a group of the formula IV.

  Suitable pharmaceutically acceptable base addition salts include, but are not limited to, metal salts such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, and also N, N′-dibenzylethylenediamine, chloro Including procaine, diethanolamine, ethylenediamine, dicyclohexylamine, meglumine (N-methylglucamine) and quaternized amines such as procaine, choline, sulfonium salts, and phosphonium salts.

  Particularly preferred compounds of the present invention are those referred to herein as SPL-7703, SPL-7304, and SPL-7320, whose structure has an active surface group consisting of 32 naphthyl disulfonic acid groups as sodium salts. Each consisting of a polylysine dendrimer, a polyamidoamine (PAMAM) dendrimer, and a polypropylene imine dendrimer scaffold. Each of the functional surface groups of the naphthyl disulfonate is attached to the branched dendrimer scaffold with an amide-methyleneoxy bond at the 32 end group.

  The present invention also provides a topical pharmaceutical composition for the prophylactic or therapeutic treatment of sexually transmitted diseases in human patients, the pharmaceutical composition comprising a compound of formula I, II, III above or The pharmaceutically acceptable salt is included with at least one pharmaceutically acceptable topical carrier or diluent.

  In another aspect, the invention also provides a method for prophylactic or therapeutic treatment of a sexually transmitted disease in a human patient, the method comprising a compound of formula I, II, III above or Including locally administering to a patient an effective amount of a pharmaceutically acceptable salt.

In another aspect, the present invention provides a compound of formula I, II, III or a medicament thereof in the manufacture of a medicament for topical administration in prophylactic or therapeutic treatment of a sexually transmitted disease in a human patient Use of chemically acceptable salts.
Detailed description of the invention

  The compounds of formula I, II, III or pharmaceutically acceptable salts thereof are novel compounds and do not anticipate exceptional activity against a broad spectrum of pathogens associated with sexually transmitted diseases Shown in

  As described above, the compounds SPL-7013, SPL-7304, and SPL-7320 are preferred compounds of the present invention and are particularly prominent against viral and microbial vectors of the most common sexually transmitted diseases. It was found to exhibit antiviral activity.

SPL-7013 has a broad spectrum of antiviral activity with high potency and minimal cellular or animal toxicity against several vectors of the most important vaginal and rectal sexually transmitted diseases. High activity is due to genital herpesvirus-2 (HSV-2), herpesvirus-1 (HSP-1) and human immunodeficiency virus (HIV-1, HIV-2) in in vitro cell tests and in vivo animal (mouse) model tests Measured in the in vitro cell test therapy. It has also been shown to be active against genital warts, pathogens causing human papillomavirus (HPV), and against bacterial vectors of nonspecific urethritis Chlamydia trachomatis. In cell tests, SPL-7013 showed activity against herpesvirus-2 resistant to currently used modified nucleoside-based antiviral agents. Furthermore, SPL-7304 and SPL-7320 are highly active against HSV-1, HSV-2, HIV-1 and HIV-2. Furthermore, SPL-7013, SPL-7304, and SPL-7320 are active in CD4-dependent and CD4-independent HIV transmission tests and are effective in preventing HIV-1 adhesion and melting. All compounds have been shown not to inhibit the growth of various species of beneficial Lactobacillus. Furthermore, SPL-7013, SPL-7304, and SPL-7320 have been shown to be effective in preventing human peripheral blood mononuclear cell infection (PBMC) by HIV-1 RoJo or SIV 89.6 pd.

  Thus, these compounds are useful in the prevention and treatment of sexually transmitted diseases as local antimicrobial agents intended for application to the vaginal or rectal mucosa to protect against sexually transmitted infections. It is.

  The present invention also provides topical pharmaceutical compositions for prophylactic and therapeutic treatment of sexually transmitted diseases in human patients, the pharmaceutical composition comprising a compound of formula I, II, III above or The pharmaceutically acceptable salt is included with at least one pharmaceutically acceptable topical carrier or diluent.

  The formulation of such compounds is well known to those skilled in the art. Suitable pharmaceutically acceptable carriers and / or diluents are any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic. And absorption enhancers or retarders, activity enhancers or retarders, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art and is described, for example, in Remington's Pharmaceutical Sciences, 18 Edition, Mack Publishing Company, Pennsylvania, USA. Except where any conventional carrier and / or diluent is not compatible with the active ingredient, its use in the pharmaceutical composition of the present invention is contemplated. Supplementary active ingredients, including agents with antiviral or antimicrobial activity, can also be introduced into the compositions of the invention.

  It is especially advantageous to formulate the composition in dosage unit form for ease of administration and uniformity of dosage. The term “dosage unit form” as used herein refers to a physically discrete unit suitable as a single dose for the human patient to be treated; each unit comprises the required pharmaceutical carrier and / or Along with the diluent is included a predetermined amount of the active ingredient calculated to obtain the desired therapeutic effect. Details regarding the novel dosage unit forms of the present invention include: (a) the unique characteristics of the active ingredient and the specific therapeutic effect to be achieved; and (b) the technology of mixing such active ingredients for specific treatments. Directed and directly dependent on inherent limitations.

  As mentioned above, the present invention also provides a method for the prophylactic and therapeutic treatment of sexually transmitted diseases in human patients, the method comprising a compound of formula I, II, III above or Including locally administering to a patient an effective amount of a pharmaceutically acceptable salt. Furthermore, the present invention relates to a compound of formula I, II, III or a pharmaceutically acceptable product thereof in the manufacture of a medicament for topical administration in prophylactic and therapeutic treatment of sexually transmitted diseases. Provide the use of salt.

  A variety of local routes of administration are available. The particular mode chosen will, of course, depend on the particular condition being treated and the dose required for prophylactic or therapeutic efficacy. Generally speaking, the method of the present invention is any mode of administration that is medically acceptable, any way of obtaining a prophylactic or therapeutic level of the active ingredient of the present invention without causing clinically unacceptable side effects. Also means. Such modes of administration include the vaginal, rectal and transdermal routes. Suitable formulations for topical, especially vaginal, rectal administration include solutions, suspensions, gels, lotions and creams, and also suppositories and microencapsulated suppositories. Other delivery systems include sustained release gels, creams, suppositories, capsules and can include sustained release delivery systems that can provide slow release of the active compounds of the present invention. Many types of transport systems are available. These include, but are not limited to, (a) an erodible system in which the active ingredient is contained in a matrix and (b) a diffusion system in which the active ingredient penetrates at a controlled rate through the polymer.

  The active ingredient of the present invention is administered in a prophylactically or therapeutically effective amount. A prophylactically or therapeutically effective amount refers to that amount necessary to at least partially achieve the desired effect, delay onset, prevent onset or progression of the particular condition to be treated, and pause. Such amount will of course depend on the particular condition to be treated, the severity of the condition, and the parameters of the individual patient including age, physical condition, size, weight, simultaneous treatment. These factors are well known to those skilled in the art and can be determined without more than routine experimentation. It is preferable to use the highest dose, ie the highest safe dose according to normal medical judgment. However, it will be appreciated by those skilled in the art that lower or acceptable doses may be administered for medical, psychological reasons, or virtually any other reason.

  In general, the vaginal or rectal dose of the active ingredient will be about 0.01 mg / kg to 1000 mg / kg per day at intervals to be determined by prevention or treatment of the disease-causing condition. Small doses (0.01-1 mg) can be administered first and then doses can be increased to about 1000 mg / kg per day. If the patient's response is inadequate at such doses, larger doses (or higher doses effective by different, more localized delivery routes) can be used to the extent that patient tolerance allows. Multiple doses per day are contemplated to achieve adequate systemic levels of the compound.

  In a particularly preferred embodiment, the present invention relates to the use of SPL-7701, SPL7304, or SPL7320 as a broad spectrum vaginal or rectal fungicide useful for the prevention and treatment of viral and microbial sexually transmitted diseases. provide. SPL-7013, SPL7304, or SPL7320 is water soluble and can be used in solution or active enhancer or retarder, enhanced or retarded absorption in topical application, adhesion to vaginal / rectal epithelium or mucosal layer Along with enhancers, they can be formulated in a suitable vehicle in the form of a gel, lotion, cream, suppository, microcapsule suspension in an aqueous or non-aqueous solvent.

  Throughout the specification and claims, unless otherwise indicated, the word “comprising” means the inclusion of the stated step, an integer, a group of integers, but the exclusion of another step, an integer, a group of integers. It will be understood that not.

Further features of the present invention will become apparent from the following examples, which are illustrative of the invention and are not intended to be limiting.

Example 1

A. BHAlyslys ₂ lys ₄ lys ₈ lys ₁₆ TFA32
1. N, N′-di-t-Boc-L-lysine (DBL)
L-Lysine HCl (1.83 kg; 10 mol) was dissolved in a solution of sodium hydroxide (880 g; 22 mol) in water (20 l) and the resulting solution was diluted with t-butanol (15). Di-t-butyl dicarbonate (4.48 kg; 20.5 mol) was added dropwise over 1 hour, during which time the reaction mixture became milky and warm (30-35 ° C.). The mixture was stirred overnight to give a clear solution. The solution was extracted with petroleum (40-60 ° C.) (2 × 10 1) and the organic layer was extracted with saturated NaHCO ₃ solution (3 × 4 1). The combined aqueous layers were cooled in an ice bath and carefully acidified to pH 2 with 1M KHSO ₄ solution. The cloudy mixture is then extracted with ether (4 × 16 l) and the combined organic layers are washed with water (2 × 8 l), dried (MgSO ₄ ), concentrated at a bath temperature of <30 ° C. and quantitatively yielded N N'-di-t-Boc-L-lysine was obtained as a very viscous oil.

2. N, N′-di-t-Boc-L-lysine-4-nitrophenyl ester (DBLONp)
A solution of dicyclohexylcarbodiamide (DCC) (2.06 kg; 10 mol) in ethyl acetate (5 l) was added di-t-Boc-L-lysine (3.46 kg; 10 mol) and 4-nitro in ethyl acetate (15 l). To an ice-cooled solution of phenol (NpOH) (1.39 kg; 10 mol) was added dropwise under nitrogen. After the addition was complete, the mixture was warmed to room temperature and stirred overnight. The resulting white suspension was then filtered and the filtrate was concentrated to give a yellow solid residue. The residue was recrystallized from ether to give N, N′-di-t-Boc-L-lysine-4-nitrophenyl ester as a white solid (yield about 60%). Many of the products could later be recovered from the mother liquor.

3. BHAlys. 2HCl
A solution of DCC (41.2 g; 0.2 mol) in dry dichloromethane (200 ml) was added to DBL (69.2 g; 0.2 mol) and benzhydrylamine (BHA) (36.65 g; 2 mol) of ice-cold solution. The mixture was stirred at 0 ° C. for 30 minutes and then at room temperature for 30 minutes. The resulting suspension was filtered and free of starting material according to the filtrate tlc (petroleum ether / EtOAc, 9: 1). The filtrate was washed with 5% HCl, water, saturated NaHCO ₃ , then dried (MgSO ₄ ) and concentrated to give a foam. Trifluoroacetic acid (TFA) (400 ml) was added to a stirred solution of foam in dry dichloromethane (200 ml). There was initially vigorous gas evolution that stopped after about 15 minutes; no starting material according to tlc (EtOAc). The solution was stirred for an additional 2 hours and then concentrated to give a brown oil. This oil was dissolved in dry acetonitrile (600 ml) and a saturated solution of HCl gas dissolved in absolute ethanol (about 360 ml) was added dropwise with swirling. A white solid immediately began to crystallize and the mixture was left for 1 hour at room temperature to complete the crystallization. The mixture is filtered and the solid is washed with dry acetonitrile, then dried and washed with BHAlys. 2HCl was obtained as a white powder (yield about 80%).

4). BHAlyslys ₂ Boc ₄
Triethylamine (39.5 ml; 0.283 mol) was added to BHAlys. In dry DMF (300 ml). To a stirred solution of 2HCl (54.4 g; 0.14 mol). A white suspension formed and was stirred for 15 minutes before DBLONp (262 g, 0.56 mol) was added dropwise. The mixture immediately turned yellow and was stirred for 3 hours, maintaining the pH of the mixture at 8-9 by dropwise addition of triethylamine. The reaction mixture was then slowly added to a large amount of vigorously stirred water and the mixture was stirred overnight. The precipitate was collected by filtration, washed with water (3x) and dried to give a yellow solid. This solid was pulverized and then washed successively with ether until it was treated with aqueous NaOH and the ether showed no yellow color. The remaining solid was dried to obtain BHAlyslys ₂ Boc ₄ as a white powder (yield about 70%).

5. BHAlyslys ₂ lys ₄ Boc ₈
Trifluoroacetic acid (600 ml) was added to a solution of BHAlys ₂ Boc ₄ (116 g; 0.12 mol) in dry dichloromethane (600 ml) and there was an immediate evolution of gas. The solution was stirred for 2 hours and concentrated to give a viscous oil. This oil was dissolved in dry DMF (500 ml) and the pH of the solution was adjusted to 8-9 with triethylamine. DBLONp (460 g; 0.99 mol) was then added and the yellow solution was stirred for 2 days at room temperature and periodic pH adjustments were made with triethylamine to maintain the pH above 8. The reaction was precipitated in water and treated as above to give BHAlyslys ₂ lys ₄ Boc ₈ as a white solid (yield about 100%).

6). BHAlyslys ₂ lys ₄ lys ₈ Boc ₁₆
Trifluoroacetic acid (1 l) was added to a solution of BHAlyslys ₂ lys ₄ Boc ₈ (200 g; 0.106 mol) in dry dichloromethane (1 l) and there was an immediate evolution of gas. The solution was stirred for 2 hours and concentrated to give a viscous oil. This oil was dissolved in dry DMF and the pH of the solution was adjusted to 8-9 with triethylamine. DBLONp (780 g; 1.67 mol) was then added and the yellow solution was stirred for 2 days at room temperature and periodic pH adjustments were made with triethylamine to maintain the pH above 8. The reaction was precipitated into water and treated as above to give BHAlyslys ₂ lys ₄ lys ₈ Boc ₁₆ as a white solid (yield about 100%).

7). BHAlyslys ₂ lys ₄ lys ₈ lys ₁₆ Boc ₃₂
Trifluoroacetic acid (1.6 l) was added to a mixture of BHAlyslys ₂ lys ₄ lys ₈ Boc ₁₆ (300 g; 0.081 mol) in dry dichloromethane (800 ml) and immediately there was a vigorous evolution of gas. The solution was stirred for 2 hours and concentrated to give a viscous oil. This oil was dissolved in dry DMF (1.2 l) and the pH of the solution was adjusted to 8-9 with triethylamine. DBLONp (1030 g; 2.21 mol) was then added and the yellow solution was stirred for 2 days at room temperature and periodic pH adjustments were made with triethylamine to maintain the pH above 8. The reaction was precipitated in water and treated as described above to give BHAlyslys ₂ lys ₄ lys ₈ lys ₁₆ Boc ₃₂ as a white solid (yield about 100%).

8). BHAlyslys ₂ lys ₄ lys ₈ lys ₁₆ TFA ₃₂
Trifluoroacetic acid (168 ml) is added to a suspension of t-butyl carbonate (Boc) protected polylysine core BHAlyslys ₂ lys ₄ lys ₈ lys ₁₆ Boc ₃₂ (20 g) in dichloromethane (350 ml) and the reaction is carried out for 14 hours at room temperature. Stir. The reaction mixture was concentrated in vacuo, dissolved in water (200 ml) and lyophilized to give BHAlyslys ₂ lys ₄ lys ₈ lys ₁₆ TFA ₃₂ (17.5 g, 83%) as a whitish solid.

B. Preparation of SPL7013 (i) [1- (oxymethylene-carboxyethyl) -3,6-naphthalenedisulfonic acid] disodium 1-hydroxy-3,6-naphthalenedisulfonic acid] disodium (100 g) was added to dimethylsulfoxide (250 ml). ) And diisopropylethylamine (60 ml) was added in one portion. Ethyl bromoacetate (38 ml) was added over 30 minutes and the reaction was stirred at room temperature for 14 hours with the exclusion of water vapor. The reaction was slowly poured into ethyl acetate (2 L) to give an oily residue / gum. The supernatant was decanted and ethyl acetate (1.5 L) was added. Washing the residue with acetone followed by several triturations gave [1- (oxymethylene-carboxyethyl) -3,6-naphthalenedisulfonic acid] disodium (97 g, 78%) as a brown solid.

(Ii) 1- (Oxymethylene-carboxy) -3,6-naphthalenedisulfonic acid NaOH aqueous solution (134 ml) in [1- (oxymethylene-carboxyethyl) -3,6-naphthalenedisulfonic acid] disodium in 500 ml of water (97 g) of solution was added all at once. The reaction was stirred for 14 hours at room temperature and then passed through an IR120 (acid form) column. The collected fractions were lyophilized to give 1- (oxymethylene-carboxy) -3,6-naphthalenedisulfonic acid (67 g, 83%) as a brown solid.

(Iii) BHAlyslys ₂ lys ₄ lys ₈ lys ₁₆ [NH—CO—CH ₂ O-3,6-naphthyl- (SO ₃ Na) ₂ ] ₃₂ —SPL7013
1- (Oxymethylene-carboxy) -3,6-naphthalenedisulfonic acid (47.85 g) was dissolved in DMF (500 ml) and diisopropylethylamine (115 ml) was added in one portion. This solution was then benzotriazol-1-yl-oxy-trispyrrolidinophosphonium hexafluorophosphate (PyBOP; 72 g) at a time with BHAlys ₂ lys ₄ lys ₈ lys ₁₆ TFA ₃₂ (15.) in DMF (650 ml). 95 g) to the solution prepared by adding to the solution. DMF (100 ml) was used to rinse the dropping funnel. The reaction mixture was stirred at room temperature for 14 hours and then diluted with water (10 L). The solution was pumped through a 1 micron deep filter across a PallFitron device (Pall Gelman) containing a 10 kD membrane. The resulting solution was passed through an IR120 (sodium form) column, pumped through a 0.22 micron deep filter and lyophilized to give SPL-7013 as a whitish solid in 75% yield. Detection wavelength set at 240 nm with a retention time of 15 minutes on an ODS-EC 15 cm × 4.6 mm HPLC column at 30 ° C. The sample was eluted at a flow rate of 1 ml per minute using the gradient elution described below.

Mass spectral analysis of SPL7013 showed a molecular weight of 16580 ± 2 daltons. Expected average molecular weight that is about SPL7013 _is 16,581.153 for _{C 583 H 577 N 64 O 287} S 64.

C. Preparation of SPL7304 In a similar manner, SPL7304 is a polyamidoamine (PAMAM) dendrimer of 1- (oxymethylene-carboxy) -3,6-naphthalenedisulfonic acid, generated 3 (Starburst, Sigma-Aldrich Pty. Ltd., Australia) And PyBOP coupling.

D. Preparation of SPL7320 In a similar manner, SPL7320 was synthesized from 1- (oxymethylene-carboxy) -3,6-naphthalenedisulfonic acid polypropyleneimind tricontamine amine dendrimer, 4.0 (DAB-Am-32, Sigma-Aldrich). Pty. Ltd., Australia) and PyBOP coupling.

Example 2
Biological activity of SPL7013, SPL7304, and SPL7320 Activity against human immunodeficiency virus The human immunodeficiency virus strains used were HIV-1 (IIIB) ¹ and HIV- (ROD) ² . Antiretroviral activity and cytotoxicity were performed in parallel. They are based on the viability of MT-4 cells infected with HIV and then exposed to various concentrations of test compound. After MT-4 cells have been grown for 5 days, the number of viable cells is determined by a colorimetric 3- (4,5-dimethylthiazol-2-yl) -2 based on tetrazolium in a 96-well microtray. , 5-diphenyltetrazolium bromide (MTT) method.

In all of these studies, virus input (multiplicity of virus infectivity, MOI) was 0.01 or 100 times the 50% cell culture infectious dose (CCID ₅₀ ). The 50% antiviral inhibitory concentration (EC ₅₀ ) was defined as the compound concentration required to protect 50% of the virus infected cells against virus cytopathicity. The 50% cytotoxic concentration (CC ₅₀ ) was defined as the compound concentration required to reduce the viability of mock-infected cells by 50%. The symbol> was used to indicate the highest concentration at which the compound was tested and found to be non-toxic. Average EC ₅₀ and CC ₅₀ values for several separate experiments were shown as defined above. In principle, individual values did not deviate more than twice or less from EC ₅₀ and CC ₅₀ values. SI is the antiviral selectivity index CC ₅₀ / EC ₅₀ .

(I) CD-4 dependent and CD-4 independent HIV transmission inhibition test methods, HIV attachment and thawing, and Lactobacillus species growth inhibitory cells and viruses:
HIV-1 _IIIB and HeLa CD4LTRβ-gal, GHOSTX4 / R5, and HL2 / 3 cell lines were obtained from the NIH AIDS Research and Reference Reagent Program (Bethesda, MD) and maintained as recommended. ME180 cells were obtained from the American Type Culture Collection (Manassas, VA).

Test sample handling and storage:
Compounds were typically dissolved in 100% DMSO and stored at −80 ° C. until testing, unless another solvent and storage conditions were specified by the sponsor. The frozen stock was thawed at room temperature, preheated at 37 ° C. for 15 minutes, and vortexed prior to preparation of the working solution in tissue culture medium. During all stages of compound dilution and handling, the compound is protected from accidental light by opaque covering and by storage and dilution in opaque or amber tissue culture plastic. Furthermore, accidental room light exposure and laminar flow tissue culture hood light exposure were adjusted by reducing total fluorescent lightning in the laboratory by 50%. The final DMSO concentration is 0.25% at the highest test concentration.

CD4-independent HIV transmission inhibition test:
ME180 cells, CD4 negative, X4 positive cervical epithelial cell lines are maintained in RPMI 1640 supplemented with 10% fetal calf serum, glutamine and antibiotics. 24 hours prior to testing, ME180 cells are trypsonized, washed and seeded in 96-well flat-bottomed microtiter plates at 2 × 10 ³ cells / well. On the day of testing, HIV-1 (H9-SKI) SKI clinical singles. Isolated chronically infected H9 cells were treated with freshly made mitomycin C (200 μg / ml) for 60 minutes at 37 ° C. This mitomycin C concentration is sufficient to cause cell death but allows viral transmission to occur. After mitomycin C treatment, H9-SK1 cells are washed three times with tissue culture medium. Test compounds are added to the ME180 monolayer followed by 2 × 10 ⁴ H9-SK1 cells. ME180 cells are co-cultured with H9-SK-1 cells and test substances for 4 hours, and H9-SK1 cells are removed by washing the ME180 monolayer three times with PBS. The wells were washed 3 times with PBS at 24 and 48 hours from the start of the test to confirm the removal of H9-SK-1 cells, and the culture was continued in a medium containing no test substance. On day 6 after co-culture, supernatants were collected and evaluated for p24 antigen expression by ELISA. Cell viability was assessed by XTT dye reduction. Compounds determined to be active in the first test were retested with or without the addition of mucin. The test in the presence of mucin is performed by adding 200 μg / ml butamtin (Sigma Chemical Co., St Louis, MO) to the transfer reaction. Transmission intervals and washings without mucin or compound replacement are performed as described above. All measurements were performed in triplicate and serial ½ Log ₁₀ dilutions of the test compound were made.

CD4-dependent HIV transmission inhibition test:
The CD4 dependent HIV transmission inhibition test was performed essentially as described for the CD4 independent transmission test, except for the use of the CD4 positive GHOST (3) X4 / R5 cell line. This cell line is derived from a HOS (human osteosarcoma) cell line that is negative for HIV co-receptor and CD4 expression. The cell line is manipulated and expressed via an HIV-2 LTR hGFP construct with T4 (CD4), R5 and X4, hygromycin selectable markers via a non-selectable retroviral vector. The cell line is as described above for the CD4 independent HIV transmission inhibition test, except that it uses 2.5 × 10 ⁴ GHOST (3) X4 / R5 and 5 × 10 ² mitomycin C-treated H9 / SK-1. Treated and cultured. Compound addition, mitomycin C treatment, and post-transmission washing to remove H9 / SK-1 were performed as described above, allowing two antiviral tests. Viral replication was assessed by measuring p24 with cells by ELISA after 3 washes 24 hours after infection to ensure a single round of infection in the presence of CD4. Compound toxicity and cell viability were assessed by XTT dye reduction. Compounds that were determined to be active in the first test were retested with or without the addition of mucin. The test in the presence of mucin is performed by adding 200 μg / ml butamtin (Sigma Chemical Co., St Louis, MO) in tissue culture medium to the transfer reaction. Transmission intervals and washings without mucin or compound replacement were performed as described above. All measurements were performed in triplicate and serial ½ Log ₁₀ dilutions of the test compound were made.

Lactobacillus test:
Lactobacillus crispatus and Lactobacillus jensenti were obtained from the American Type Tissue Culture Collection and grown in Lactobacilli MRS broth (Difco, Fisher Scientific, Pittsburgh, PA). This medium allows for sufficient growth of Lactobacilli under anaerobic conditions. Bacillus stocks were made and frozen in 15% glycerol at −80 ° C. for use in susceptibility testing. L. crispatus and L. To assess the effect of compounds on jensenti growth, 10 ml of MRS medium is inoculated with a stub from a glycerol bacterial stock. Cultures were placed in Gas Pak CO ₂ bags and incubated at 37 ° C. for 24 hours. The next day, the Lactobacillus culture is diluted to an OD of 0.06 at a wavelength of 670 nm. The compound was diluted and placed in a 96 well flat bottom plate and Lactobacillus sp. Was added. Commercially available penicillin / streptomycin solutions at high test concentrations of 1.25 U / ml and 1.25 μg / ml, respectively, were used as positive controls. Plates are again incubated for 24 hours at 37 ° C. in a Gas Pak CO ₂ bag, and bacterial growth is measured by optical density measurement at 490 nm in a molecular device reader. All measurements were performed in triplicate and 6 1/2 Log ₁₀ dilutions were made from the high test concentration.

Virus adhesion test:
This test was designed to detect compounds that interact with cells and block virus attachment and / or interact with the adhesion / melt complex that forms. The adhesion test is performed using HeLa CD4 LTR βgal cells. HeLa CD4 LTR βgal cells are routinely cultured with the necessary selective antibiotics. Twenty-four hours before the start of the test, the cells are trypsinized, counted, and 1 × 10 ⁴ cells are placed in 0.2 cm wells in medium without selective antibiotics. The medium is removed at 24 hours and the compound in the medium is placed on the cells and incubated at 37 ° C. for 15 minutes. A known titer of HIV strain IIIB is then added to the wells and incubation is continued for 1 hour. At the end of the culture, the wells are washed 3 times with medium and the culture is continued for 40-48 hours. At the end of the test, the medium is removed and β-galactosidase enzyme expression is determined by the manufacturer's instructions by a single-step chemiluminescent method using a single solution to lyse cells and detect β-galactosidase enzyme activity. Measured by chemiluminescence according to (Tropix, Gal-screen, Bedford Mass.). Compound toxicity is monitored on sister plates by XTT dye reduction. All measurements are performed in triplicate with serial 1/2 Log dilutions of the test material. The 1 hour viral adsorption interval is short enough that AZT, which requires phosphorylation to its active triphosphate form (AZZZ-TPP), is not active in this test.

Melting test:
The melting test evaluates the ability of a compound to block cell-cell melting mediated by HIV-1 Env and CD4 expressed in another cell. This test is sensitive to both gp120 / CD4 interactions and inhibitors of X4 coreceptors. First, 5 × 10 ³ HeLa CD4 LTR βgal cells are placed in macrotiter wells and cultured overnight. The next day, media is removed and HeLa CD4 LTR βgal cells are incubated for 1 hour at 37 ° C. with fresh media with test compounds. After incubation, 5 × 10 ³ HL2 / 3 cells are added and incubation is continued for 40-48 hours. At 40-48 hours, β-galactosidase enzyme expression is detected by chemiluminescence (Tropix, Gal-screen, Bedford Mass.). Compound toxicity is monitored on sister plates using XTT dye reduction. All measurements are performed in triplicate 1/2 Log dilutions of the test material in triplicate.

p24 antigen ELISA:
ELISA kits are obtained from Coulter Electronics and detection of supernate or cell associated p24 antigen is performed according to manufacturer's support. For cell-associated p24, cell lysates are prepared by lysing well contents in 25-50 μl of Coulter-fed virus lysis buffer and tested after one round of freeze / thaw. All p24 measurements are made after serial dilution of the sample to confirm the absorption of the linear portion of the standard p24 antigen curve. Standard curves are manufactured using standard products and instructions supplied by the manufacturer. Data are expressed in pg / mlp24 antigen, with final concentrations calculated from optical density using Molecular Devices Soft Max software package.

XTT staining for cell viability and compound toxicity:
The TC ₅₀ for the test substance is the tetrazolium dye XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5 [(phenyl) in replicate microtiter plates containing cells and compounds without virus. Derived by measuring the reduction of amino) carbonyl] -2H-tetrazolium hydroxide). XTT is metabolized to a soluble formazan product by the mitochondrial enzyme NADPH oxidase in metabolically active cells, allowing rapid quantitative analysis of cell viability and compound toxicity. XTT solution is prepared daily as a 1 mg / ml stock in PBS. Phenazine methosulfate (PMS) solution is prepared at 15 mg / ml in PBS and stored at −20 ° C. in the dark. XTT / PMS stock is prepared immediately prior to use by diluting PMS 1: 100 in PBS and adding 40 μl / mL XTT solution. 50 μl of XTT / PMS is added to each well of the plate and the plate is incubated at 37 ° C. for 4 hours. It was experimentally determined that the 4 hour incubation was within the linear response range for MTS dye reduction, with the indicated number of cells for each test. An adhesive plate sealer is used in place of the lid, the sealed plate is inverted several times to mix the soluble formazan product, and the plate is read at 450 nm using a Molecular Devices Vmax 96 well plate format spectrophotometer.

^１ペニシリンおよびストレプトマイシンの出発濃度はそれぞれ１．２５U/mlおよび１．２５μｇ／ｍｌである

Data Analysis Dextran sulfate (positive) and dextran (negative) are used as controls for CD4-dependent and CD4-independent KIV transmission inhibition tests. The sulfonic acid dye Chicago Sky Blue is used as a positive control for adhesion and melting tests ⁴ . Commercially available penicillin (10,000 U / ml) streptomycin (10.0 mg / ml) solution is used as a positive control for the Lactobacillus sensitivity test. Where appropriate, for each compound, an IC ₅₀ (concentration that inhibits viral replication or transmission by 50%), ID ₅₀ (concentration that inhibits 50% growth of Lactobacillus) TC ₅₀ (concentration that results in a 50% reduction in cell viability) And therapeutic index (TI: TC ₅₀ / IC ₅₀ or ID ₅₀ ) is calculated by linear regression

The starting concentrations of ¹ penicillin and streptomycin are 1.25 U / ml and 1.25 μg / ml, respectively.

(Ii) Activity of SPL7013, SPL7304, and SPL7320 against HIV-1 RoJo and SIV89.6pd
Method
Viral human immunodeficiency virus type 1 (HIV-1) strain RoJo is a low-passage pediatric isolate derived from the laboratory of the Southern Research Institute (SRI). SHIV 89.6 pd was obtained from Mark Lewis of SRI and stocks were grown in human PBMCs for antiviral testing.

PBMC isolation and blasting peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll hyper-gradient separation from normal hepatitis and HIV-1 negative donors. Simply put. Anticoagulated blood was diluted 1: 1 with Dulbecco's phosphate buffer (PBS), excluding Ca ⁺⁺ and Mg ⁺⁺ , and overlaid on 14 ml of lymphocyte separation medium in a 50 ml centrifuge tube. The tube was then centrifuged at 600 xg for 30 minutes. Banded PBLs were gently aspirated from the resulting interface and subsequently washed twice with PBS by low speed centrifugation. Mononuclear cells were counted, viability was measured by Trypan Blue dye exclusion, 15 μF FBS (heat inactivated), 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin and 2 μg of 10 μg / mL gentamicin Resuspended at 1 × 10 ⁶ cells / mL in RPMI 1640 supplemented with / mL phytohemagglutin (PHA). Cells were cultured for 48-72 hours at 37 ° C., 5% CO ₂ . After incubation, the cells were collected by centrifugation, washed, and RPMI 1640 supplemented with 15% FBS (heat inactivated), 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin and 10 μg / mL gentamicin. Resuspended with 20 U / mL recombinant IL-2 (R & D Systems, Minneapolis, Minn.). IL-2 was included in the culture medium to maintain cell division initiated by PHA mitogen stimulation. The cells were cultured in IL-2 for 72 hours and then used for virus tuning.

Human peripheral blood mononuclear cells from a minimum of 2 donors blasted with PBMC test PHA and IL-2 were counted and viability was measured by Trypan Blue dye exclusion and mixed in equal proportions. Pooled donors were used to minimize the variation observed between individual donors resulting from quantitative and qualitative differences in HIV infection and the overall response of primary lymphocytes to PHA and IL-2. Cells are supplemented with 15% FBS (heat inactivated), 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μg / mL gentamicin and IL-2 (20 U / mL, R & D Systems, Minneapolis, Minn.) Resuspended at 1 × 10 ⁶ cells / mL in RPMI 1640 without phenol red. 50 μL of cells were then dispensed into the internal 60 wells of a 96 well round bottom microtiter culture plate in a standard format developed by the Infectious Disease Research department of Southern Research Institute. Each plate contains a cell control well (cells only), a virus control well (cell plus virus), and an experimental well (drug plus cell plus virus). Serially diluted compounds were added to the microtiter plate, and appropriate pretiter strains of HIV-1 and SHIV-1 were added. All samples were tested in triplicate on virus-free replicate plates for determination of compound toxicity. The final volume per well was 200 μL. The test was incubated for 6 days in a humidified atmosphere at 37 ° C., 5% CO ₂ , after which the supernatant was collected for analysis of RT activity and the sister plates were analyzed for cell viability by MTS dye reduction. The wells were examined microscopically and any abnormalities were noted.

At the end of the MTS staining test for cell viability , test plates are stained with the soluble tetrazurium-based dye MTS (CellTiter Reagent Promega, Madison, Wis.) To determine cell viability and quantify compound toxicity Turned into. MTS is metabolized to soluble formazan products by metabolically active cellular mitochondrial enzymes, allowing rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a single stable solution that does not require preparation prior to use. At the end of the PBMC test, 20 μL of MTS reagent was added to the wells and the wells were incubated at 37 ° C. for 4 hours. An adhesive plate sealer was used instead of the lid, the sealed plate was inverted several times to mix the soluble formazan product, and the plate was read spectrophotometrically on a Molecular Devices Vmax plate reader.

Reverse transcriptase test Reverse transcriptase activity was measured in supernatants without cells. Tritiated thymidine triphosphate (NEN) (TTP) was resuspended in distilled water at 5 Ci / ml. Poly rA and oligo dT were prepared as stock solutions and kept at -20 ° C. RT reaction buffer was made fresh daily, 125 μL 1.0 M EGTA, 125 μL dH ₂ O, 110 μL 10% SDA, 50 μL 1.0 M Tris (pH 7.4), 50 μL 1.0 M DTT and 40 μL Of 1.0M MgCl ₂ . These three solutions were mixed in a ratio of 2 parts TTP, 1 part poly rA: oligo dT, and 1 part reaction buffer. 10 μL of this reaction mixture was placed in a round bottom microtiter plate and 15 μL of supernatant containing virus was added and mixed. Plates were incubated at 37 ° C. in a water bath with a sturdy support to prevent plate flooding and incubated for 60 minutes. After the reaction, the reaction volume was spotted on a piece of DE81 paper, washed 5 times 5 minutes with 5% sodium phosphate buffer, 2 times 1 minute distilled water, 2 times 1 minute 70% ethanol, and then dried. Opti-Fluor O was added to each sample, introduced, and radioactivity was quantified using a Wallac 1450 Microbetaplus liquid scintillation counter.

Data analysis gives IC ₅₀ (50% inhibition of viral replication), TC ₅₀ (50% reduction in cell viability) and therapeutic index (TI, IC ₅₀ / TC ₅₀ ). AZT was used as the relevant positive control compound for each test. The results of the antiviral evaluation performed are summarized in the following table.

B. Activity against herpesvirus-HSV-1 and HSV-2 (i) Virus plaque reduction test General methods HSV standard strains G (HSV-2) and F (HSV-1) were used in the test. The virus input for 6 well plates was 100 pfu / well. An HSV sensitive cell line, Vero cells, was used in the virus production reduction test. An epithelial cell line, Hela-229 cells, was used for the cytotoxicity test.

  The antiviral effect of the compounds was measured by a modified plaque reduction test. Confluent cells were washed with PBS and subsequently infected with HSV (100 pfu / well) for 1 hour at 37 ° C. Tilt every 10 minutes. After removing the virus inoculation, the infected cells were washed with PBS and covered with 0.5% methylcellulose in culture medium (equal volume of 1% methylcellulose mixed with 2x culture medium). Cells were incubated at 37 ° C. for 2 days for HSV-2 infection and 3 days for HSV-1 infection. When the plaque size was appropriate, the cells were fixed with 10% formalin for 10 minutes. The plaques were then stained with 0.5% crystal violet for 10 minutes. The dye was removed by washing with tap water and left to dry in a fume hood. The plaques were then counted.

  All data was obtained from two experiments. The average plaque count in the test well was compared to the average plaque count in the control well.

The EC ₅₀ (concentration giving a 50% reduction in inoculum plaque count) was calculated. Antiviral activity and cytotoxicity measurements were performed in parallel on the same strain of cells. 50% cytotoxicity (CC ₅₀ ) was defined as the compound concentration required to reduce the viability of mock-infected cells by 50%.

Infection Pretreatment Method Culture medium was removed from confluent Vero cells in 6-well plates and washed with 1 ml PBS. 1 ml of culture medium containing compounds at concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1.3, 10, 30 μg / ml was added to each well and incubated at 37 ° C. for 1 hour. . After preincubation of the cells with dendrimers, the cells were then infected with 100 pfu / well of HSV-1 or HSV-2. Infections were incubated at 37 ° C. for 1 hour and tilted every 10 minutes. After removing the ingestion, the infected cells were coated with 1.5 ml of 0.5% methylcellulose diluted in 2 × culture medium for plaque testing.

Treatment of infected cells Confluent Vero cells were washed with PBS. Cells were then infected with 100 pfu / well of HSV-1 or -2 for 1 hour at 37 ° C. After removal of the virus inoculum, the infected cells are washed once with PBS and contain dendrimers at concentrations of 0, 0.03, 0.1, 0.3, 1.3, 10, 30, and 100 μg / ml Coated with 0.5% methylcellulose. Cells were incubated for 2 day (HSV-2) or 3 day (HSV-1) plaque assay at 37 ° C.

Dendrimer Cytotoxicity Culture media was removed from confluent HeLa-229 cells in 24-well plates by a pump. The cells were then washed once with 1 ml PBS. 1 ml of culture medium containing dendrimer at concentrations of 0, 100, 500, and 1000 μg / ml was added to each well. The cells were incubated for 2 days in a 37 ° C. incubator. At the end of the incubation, the medium was removed and the cells were washed with PBS. 500 μl of 0.01% neutral red (in PBS) was then added to each well and incubated at 37 ° C. for 30 minutes. The dye was then removed and the cells were washed twice with 1 ml PBS per well. The dye was extracted by adding 500 μl of 50% ethanol in PBS and 1% glacial acetic acid to each well and incubated at room temperature for 15 minutes with gentle shaking at 120-150 rpm. 100 μl dye extracted from each well was placed in a 96-well plate and the absorbance at 550 nm was read with an ELISA reader.

(Ii) Herpesvirus-2 efficacy in animal models
The efficacy of SPL-7013 was tested in a genital HSV prevention test in a mouse (strain MS) model. A 15 μl dose of 100 mg / ml of compound or 10 mg / ml was dropped into the vagina of 16 animals 20 seconds prior to HSV-2 infection. SPL-7013 prevented infection and used the same number of all-day mortality at 3 days of control infected mice as compared to the control disease of all animals tested compared to the control.

C. Human Papillomavirus (HPV) Inhibition of Human Epithelial Cell Uptake Compounds were evaluated for their ability to inhibit the binding and uptake of human papillomavirus-like particles to the human epithelial cell line.

  The compounds were tested in the range of 6 dilutions as binding inhibitors and uptake inhibitors of papillomavirus fluorochrome-labeled virus-like particles (VLPs) of human papillomavirus type 6b. VLPs were bound to and taken up into epithelial cells in the presence of SPL-7013. Binding and uptake studies were measured using flow cytometry, and inhibition of binding was reported as a percentage of the observed binding in the absence of inhibitor. The test was conducted in two independent tests.

Way cells. Human epidermoid carcinoma cell line A431 was purchased from American Type Culture Collection with Passage 30 (CRL-1555, Batch F-13530) and stored in DMEM in 10% FCS.

  Human papillomavirus type 6b VLPs were grown and purified according to standard operating procedures. The particles were labeled with fluorochrome.

  The compound was redissolved in sterile water to a concentration of 5 mM, used fresh for the first test, and then frozen at −20 ° C. It was then thawed for the second test.

Uptake test A T431 flask of A431 cells was washed with 10 ml PBS / EDTA (0.05%) for 5 minutes at 37 ° C and then treated with 2 ml trypsin for an additional 5 minutes at 37 ° C. DMEM / 10% FCS (10 ml) was added to the cells, centrifuged at 1000 × g for 5 minutes at room temperature, and resuspended in complete medium at 3 × 10 ⁵ cells / ml in a 15 ml tube. The cells were incubated at 37 ° C. for 2 hours with inversion every 20 minutes to allow cell surface protein re-expression. Cells were centrifuged at 1000 × g for 5 minutes at room temperature, resuspended at 3 × 10 ⁵ cells / 100 μl in serum free DMEM, and 100 μl of suspension was placed in a 1.5 ml tube. Compounds were added to the cells at the next 6 dilutions in 10 μL PBS. 100 μM, 10 μM, 1.0 μM, 100 nM, 10 nM, 1.0 nM.

  Cells were incubated with compounds at 37 ° C. for 30 minutes before adding labeled VLPs (200 ng). As a positive control, only VLPs were added to the cells, and the negative control was VLPs added, but cells incubated on ice. Cells were mixed and incubated for 2 hours at 37 ° C. Cells were washed once with 1 ml PBS and fixed in FACS buffer (P13S / 4% paraformaldehyde). Analysis was performed on a Coulter FACS machine.

The analysis results were analyzed using WinLisT. First, the cell size was analyzed using a front / side scatter, and live cells were gated. The average fluorescence intensity (MFI) of this gate was made and used for further analysis. Uptake was reported as a percentage of the binding observed for VLP (100%) only to cells with VLPs incubated with ice (0%).

Effect SPL-7013. Maximum inhibition of uptake (26% uptake) was observed at 1 μM with no further inhibition at higher concentrations. This compound still showed inhibition of uptake at 1 μM and 10 nM. Virus uptake increased after this point with a decrease in SPL-7013 concentration.

D. Chlamydia trachomatis infection Efficacy of SPL-7013 in an animal model Twenty seconds prior to infection with Trachomatis, they were treated in the upper or lower genital tract with a 15 μL drop of a 100 mg / mL solution of SPL-7013N. Lower genital tract infection is defined by isolation of organisms by culture from vaginal swab samples collected 3 or 6 days after challenge. For upper genital tract infection, the definition is the isolation of organisms by culture from upper genital tract tissue collected 10 days after challenge. Mice treated with phosphate buffer PBS were used as controls.

Those skilled in the art will appreciate that changes and modifications other than those specifically described may be made to the invention as broadly described herein without departing from the spirit and scope of the invention. It will be understood that the invention extends to include all such changes and modifications.

Claims (10)

A compound of formula I, II, or III or a pharmaceutically acceptable salt thereof.

In which R represents a group of the formula IV.

  2. A compound according to claim 1 wherein the pharmaceutically acceptable salt is selected from metal salts, organic salts with organic amines, or quaternized amines, sulfonium salts and phosphonium salts.   The compound of claim 2 wherein the metal salt is an aluminum, calcium, lithium, magnesium, sodium or zinc salt.   The compound of claim 2, wherein the organic salt with an organic amine is N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethylenediamine, dicyclohexylamine, meglumine (N-methylglucamine) or procaine.   The compound of claim 2 wherein the quaternized amine is choline. Compound of formula I

(Wherein R represents a group of formula IV)

Or a pharmaceutically acceptable salt thereof. Compound of formula II

(Wherein R represents a group of formula IV)

Or a pharmaceutically acceptable salt. Compound of formula III

(Wherein R represents a group of formula IV)

  A topical pharmaceutical composition for prophylactic and therapeutic treatment of HSV-1, HSV-2, HIV-1, HIV-2, HPV infection or Chlamydia trachomatis in a human patient, comprising: A composition comprising a compound according to any one together with at least one pharmaceutically acceptable topical carrier or diluent.   9. In the manufacture of a medicament for topical administration in preventive and therapeutic treatment of HSV-1, HSV-2, HIV-1, HIV-2, HPV infection or Chlamydia trachomatis in a human patient. Use of a compound according to any one.